Chemotaxis of vascular smooth muscle cells (VSMCs) is integral to the development of atherosclerosis and post-angioplasty restenosis. We have shown that chemotaxis of cultured rat VSMCs in a Boyden chamber is dependent on the gradient of the chemoattractant, platelet-derived growth factor (PDGF), and occurs infrequently and asynchronously among cells (only 10% chemotaxis within 4 hrs). We examined whether intracellular calcium (Cai) signaling mediates chemotaxis and could contribute to the asynchronous migratory response. In order to evaluate Cai in individual migrating VSMCs, we adapted a Boyden chemotaxis chamber to an imaging microscope which allows simultaneous measurement of locomotion and Cai in VSMCs (loaded with the Cai indicator, fura-2) during chemotaxis. There are two distinct, sequential cellular Cai signaling phases in response to a PDGF gradient: (1) an initial phase (time frame of minutes), with a relatively synchronous, transient increase in Cai in most cells, and (2) a delayed phase (time frame of hours), with asynchronous increases in Cai among individual cells which precede chemotaxis. The Cai achieved by migrating VSMCs was significantly higher compared to non-migrating VSMCs (mean Cai : 630 plus/minus 33 nM and 266 O 18 nM, respectively). Neither chemotaxis nor late increases in Cai were observed in VSMCs in the absence of PDGF or of its gradient. The PDGF gradient, which apparently conveys information that uniform- concentration exposure does not, is necessary for the late phase Cai responses in migrating cells. Buffering the late increase (but not the early increase) in Cai with intracellularly-loaded BAPTA significantly attenuated chemotaxis. The late Cai increase which occurs during chemotaxis results, in part, from trans-sarcolemmal Ca(2+) influx, as chemotaxis varied directly with extracellular Ca(2+). This Ca(2+) influx occurs through non-L-type Ca(2+) channels. In migrating VSMCs, the late Cai increase is coincident with activation of calcium/calmodulin-dependent protein kinase II (CaMK II), requisite during VSMC chemotaxis, which may facilitate essential cytoskeletal remodeling. Thus, changes in Cai mediate VSMC chemotaxis, and differences in Cai signaling within individual cells contributes to the asynchronous chemotactic response within VSMC populations.